Carotenoid is a pigment which is widely distributed in the animal and plant kingdoms, and is classified as tetraterpene (C40) having isoprene C5H8 as a structural unit. Examples of carotenoids include α-carotene, β-carotene, γ-carotene, lycopene, cryptoxanthin, lutein, zeaxanthin, canthaxanthin, fucoxanthin, antheraxanthin, violaxanthin and the like. Carotenoids include a substance which may be converted within the animal body to vitamin A (provitamin A), and a substance having high anti-oxidant action is also known.
Among these carotenoids, β-cryptoxanthin contained in citrus fruits, such as Satsuma mandarin (Citrus unshiu Marc.) and Valencia orange, loquat, persimmon, peach, red bell pepper, papaya and the like clearly has not only characteristics as provitamin A but also high anticarcinogenic action and high anti-oxidant action. Hence, β-cryptoxanthin has drawn great attention in recent years.
β-Cryptoxanthin tends to be affected by light, heat and oxygen; β-cryptoxanthin tends to be isomerized (converted into a cis isomer) by light and heat and, furthermore, polyene is cleaved by oxidation. There have been only few studies on a method for the easy and accurate measurement of the amount of β-cryptoxanthin contained in fruit juice or the like.
Non-patent Document 1 describes a method of quantitatively determining β-cryptoxanthin contained in Satsuma mandarin juice. Specifically, in the method described in Non-patent Document 1, first 1 g of diatomaceous earth and 10 ml of ethanol are added to 10 g of fruit juice and stirred, and then the mixture is poured onto a thin bed of diatomaceous earth on a glass filter, and washed with ethanol until the color of residues disappears, whereby an ethanol extract is obtained. This ethanol extract (about 60 ml) is then transferred by washing to a separatory funnel. Then, equivalent amounts of ether and distilled water are added, and a gas layer is replaced with nitrogen gas, followed by shaking to distribute carotenoid to the ether. This ether solution is concentrated in vacuo, and an equivalent amount of 10% methanolic KOH solution is added. The mixture is allowed to stand at 20° C. in a dark place under a nitrogen atmosphere for 1 hour to conduct saponification treatment. An unsaponifiable material is fractionated with ether from the solution having been subjected to the saponification treatment, and is washed with distilled water until an alkali is removed. This is followed by dehydration with anhydrous sodium sulfate and removal of the solvent by distillation in vacuo under a nitrogen atmosphere. Thereafter, a mobile phase solvent is added to give a volume of 10 ml, and a solution having been passed through a 0.45-μm membrane filter is analyzed by an HPLC apparatus equipped with a C30 analysis column. A calibration curve is formed using a sample prepared similarly using a β-cryptoxanthin standard specimen, and quantitative analysis of β-cryptoxanthin in the fruit juice is conducted.